Section7:Batch Co-Transfection and Cell Banking Protocols

From Assay Guidance Wiki

The use of transient transfection and co-transfections in cell based assays for screening compounds tends to have higher variability than non transfected cell based assays. The process of scaling up the cells and the transfection process also adds days to the process of the assay. By banking the transfected or co-transfected cells the process becomes less variable and more efficient. Below is the protocol for batch co-transfection and cell banking.

1. Counting Cells for Transfection
   • Allow both the 0.05% Trypsin and the Assay media to warm up at room temperature prior to detaching cells.
   1. Remove the flask out of the incubator (37°C and 5% CO2) that will be used for counting.
   2. Aspirate off the media with an aspirating pipette attached to the vacuum source located within the culture hood.
   3. Add 10mls of Dulbecco’s PBS to each flask. Next, rock the flask(s) back and forth once making sure to wash the side of the flask with the cells attached.
   4. Aspirate off PBS.
   5. Add 3 ml of 0.05% Trypsin/flask. Rock the flask(s) back and forth to coat the cells with the Trypsin.
   6. Let sit at room temperature for 3 minutes. Whack flask to detach cells.
   7. Add 7mls media to flask to quench the Trypsin. Pipette up and down several times to break up clumps.
   8. Transfer the cell mixture to a 50 ml blue-top centrifuge tube and mix well.
   9. Count the cells three times using a Coulter cell counter and calculate the average.
   10. Next, calculate the number of cells using the following formula:
       
       (Avg. #of counts)/(vol. the counter will count, µl) X (vol. of isoton, µl )÷(vol. of the sample, µl) X (total vol. of the cells sampled from, µl)
       Example: 6279 counts/500 µl X 20,000 µl ÷ 100 µl X 10,000 µl = 25.11 million
2. Transfections
   1. Prepare the Serum-free media and transfection reagent mix in a 250 ml orange-top conical centrifuge tube according to the optimized ratio for transfection. Mix gently halfway through and after adding all of the reagent and incubate for 5 minutes. DO NOT touch transfection reagent to the plastic sides of the tube. Dispense directly into the SFM.
   2. Add appropriate amounts of DNA to the tube with the SFM and transfection reagent mix. Mix, by tapping the tube gently after additions.
   3. Incubate for 30 minutes at room temperature.
   4. During this incubation period, aspirate media from the T225 flasks to be transfected and add back 38mls of Assay Media. Place back in the incubator at 37°C, 5% CO2.
3. Cell Banking
   • Allow both the 0.05% Trypsin and the Assay media to warm up at room temperature prior to detaching cells.
   1. Remove the flasks out of the incubator (37°C and 5% CO2).
   2. Aspirate off the media with an aspirating pipette attached to the vacuum source located within the culture hood.
   3. Add 10mls of Dulbecco’s PBS to each flask. Next, rock the flask(s) back and forth once making sure to wash the side of the flask with the cells attached.
   4. Aspirate off PBS.
   5. Add 3 ml of 0.05% trypsin/flask. Rock the flask(s) back and forth to coat the cells with the Trypsin.
   6. Let sit at room temperature for 3 minutes. Whack flask to detach cells.
   7. Add 7mls media to flask to quench the trypsin. Pipette up and down several times to break up clumps.
   8. Transfer the cell mixture to a 250 ml orange-top centrifuge tube and mix well.
   9. Divide cell suspension between orange-top tubes.
   10. Count the cells three times using a Coulter cell counter and calculate the average.
   11. Next, calculate the number of cells using the following formula:
       
       (Avg. #of counts)/(vol. the counter will count, µl) X (vol. of isoton, µl )÷(vol. of the sample, µl) X (total vol. of the cells sampled from, µl)
       Example: 6279 counts/500 µl X 20,000 µl ÷ 100 µl X 10,000 µl = 25.11 million
       
       
   12. Spin tubes in centrifuge at 1500 RPM’s for 5 minutes.
   13. Remove supernatant.
   14. Divide total number of cells (see #9) by 50 (cell concentration/ vial frozen: 50X10e6) = mls freezing solution to add to cell pellet.
   15. Pipette up and down several times to break up cell clumps.
   16. Aliquot 1 ml of cell suspension to a 2 ml cryogenic vial.
   17. Place vials in a Mr. Frosty and place in -80°C freezer for approximately 4 hours.
   18. Remove and place in Liquid Nitrogen tank for long term storage.
       
       Freezing Solution
       • 90% Fetal Bovine Serum (Charcoal Stripped FBS)
       • 10% DMSO
4. Validation of Frozen Cells
   1. Remove vial from liquid nitrogen storage.
   2. Thaw quickly in 37°C water bath.
   3. Bring up slowly to total volume of 20-30 mls of cold Assay media.
   4. Spin at 1500 RPM’s for 5 minutes. Remove supernatant.
   5. Re-suspend in 10 mls assay media. Take a 100 ul aliquot of cell suspension and add to 20 mls Isoflow.
   6. Count the cells three times using a Coulter cell counter and calculate the average cell count.
      
      (Avg. #of counts)/ (vol. the counter will count, µl) X (vol. of isotonic, µl) ÷(vol. of the sample, µl) X (total vol. of the cells sampled from, µl)
      Example: 6279 counts/500 µl X 20,000 µl ÷ 100 µl X 10,000 µl = 25.11 million
      
      
   7. Seed a 96 well or 384 well microtiter plate following one of the below equations to determine cell number in suspension.
      
      384 well format (30,000 cells in 50 ul):
      1. (11.5 mill. Cells/plate) (_____plates, included 2 extra) =_____mill. Cells needed
      2. (____mill. Cells needed for assay accounting for extra) / (____mill. cells/ml) = ______ml of cells from cell stock
      3. (____mill. cells/ml) (____ml of cells from cell stock) / (0.6 mill. cells/ml) = ml total
      4. (____ml total) – (____ml of cells from cell stock) = ml of media

      96 well format (50,000 cells in 80 ul):

      1. (4.8 mill. Cells/plate) (____plates, included 1 extra) = ____mill. Cells needed
      2. (____mill. Cells needed for assay accounting for extra) / (____mill. cells/ml) = ____ml of cells from cell stock
      3. (____mill. cells/ml) (____ml of cells from cell stock) / (0.625) mill. cells/ml) = ml total
      4. (____ml total) – (____ml of cells from cell stock) = ml of media
      
      
   8. Incubate overnight overnight in 37°C, 5% CO2
   9. Dose with appropriate dose response curve for each receptor